1. Field of the Invention
The present invention generally relates to methods of microencapsulating bioactive substances, and particularly to methods utilizing interfacial coacervation at the immiscible interface of two liquid phases. More particularly, the invention pertains to such methods which maintain conditions of low shear force during formation of the microcapsules. The present invention also pertains to microcapsules formed by such methods and to their methods of use.
2. Description of the Prior Art
Liquid microcapsules and liposomes are often used to store and deliver bioactive substances such as drugs, enzymes or biocatalysts. One recent effort to provide liposomes with enhanced circulation times is that disclosed in U.S. Pat. No. 5,013,556 to Woodle et al. Liposomes created by Woodle et al. contain 1-20 mole % of an amphipathic lipid derivatized with a polyalkylether (such as phosphatidyl ethanolamine derivitized with polyethyleneglycol). Another improvement is provided by U.S. Pat. No. 5,225,212 (issued to Martin et al.) which discloses a liposome composition for extended release of a therapeutic compound into the bloodstream. Those liposomes are composed of vesicle-forming lipids derivatized with a hydrophilic polymer, wherein the liposome composition is used for extending the period of release of a therapeutic compound such as a polypeptide, injected within the body. Formulations of xe2x80x9cstealthxe2x80x9d liposomes have also been created with lipids that are less detectable by immune cells in an attempt to avoid phagocytosis (Allen et al. (1992) Cancer Res. 52:2431-39.) Still other modifications of lipids (i.e., neutral glycolipids) may be made in order to produce anti-viral formulations. U.S. Pat. No. 5,192,551 to Willoughby et al. 1993. However, new types of liposomes and microcapsules are needed to exploit the various unique applications of this type of drug delivery.
Many proteins of interest, such as those containing bioactive drug sites or enzymatically active sites, are only slightly soluble in aqueous solutions, which limits the quantity of drug that can be microencapsulated by usual techniques. In an effort to increase the amount of drug delivered to the target tissues, crystalline drug suspensions are sometimes encapsulated. Fragile liposome or non-lipid carriers too often rupture or are pierced by the sharp crystals, however, leading to loss of the drug before it reaches its target. This undesired release of the drug crystals has also been known to damage the lining of blood vessels.
Others have endeavored to increase the amount of drug in a liposome by loading the drug into the liposome by via a pH gradient. U.S. Pat. No. 5,192,549 (issued to Barenolz and Haran) describes methods for forming liposomes and then obtaining transmembrane loading of amphiphatic drugs into the liposomes using an ammonium ion gradient between the internal and external aqueous phase on either side of the liposome membrane. The movement of ammonium from inside the liposome to the outside causes a pH change inside, thereby creating a driving force for the amphiphatic drug to be loaded or released through the membrane. Disadvantages of this method are that it requires the encapsulation of ammonium sulfate or another ammonium salt inside the liposomes, and transmembrane transport is limited to weak amphiphatic compounds. This type of drug concentrating method has not been used successfully to form encapsulated crystals, however. If this method were applied to protein crystal growth inside the liposome, it would be limited to applications where the protein was compatible with the ammonium salts and dissolved NH4.
Another area where protein crystals are used is in macromolecular crystallography, which requires large, high-quality protein crystals. Conventional methods of growing protein crystals, as required for x-ray diffraction studies of three-dimensional structure, are often compromised by the formation of multiple small crystals, amorphous precipitates and aggregates rather than a single, or a few, large crystals from the limited amount of protein in the available mother liquor. It has been estimated that about 1015 molecules are required to make up a crystal of sufficient size for x-ray crystallographic examination (Proteins Structures and Molecular Properties, 2nd Ed., Thomas E. Creighton, Ed., W.H. Freeman and Co., NY, N.Y., p. 203). It is often observed that, with conventional techniques, the best crystals begin to redissolve because of fluid perturbations at the crystal surface, temperature shifts and other changes in the mother liquor surrounding the crystal. Carrier fluids used to wash the crystal free from the mother liquor or used during mounting of the crystal (for x-ray diffraction) also tend to cause redissolution of the crystal before it can be analysed.
There are many existing methods aimed at enhancing protein crystal growth, some of which take advantage of the favorable crystal growing conditions found in microgravity. An apparatus for carrying out crystallization of proteins and chemical syntheses by liquid-liquid diffusion in microgravity is described in U.S. Pat. No. 4,909,933 (issued to Carter et al.) Another apparatus, disclosed in U.S. Pat. No. 5,130,105 (issued to Carter et al.) relies on vapor diffusion growth of protein crystals. Other recent microgravity-dependent methods are disclosed in U.S. Pat. No. 5,106,592 (issued to Stapelmann et al.), which deal with hanging drop vapor diffusion, dialysis of the protein solution, and interface diffusion between the protein solution and a precipitating agent.
A ground-based (i.e., Earth normal gravity) method of concentrating protein solutions to obtain crystal growth is described by Todd et al. ((1990) J. Crystal Growth 110: 283-292), and U.S. Pat. No. 5,104,478 (issued to S. K. Sikdar et al.), which relies on osmotic dewatering of protein solutions. Todd et al. and Sikdar et al. describe the use of a dual chamber device wherein a near-saturated protein solution is separated from a highly osmotic solution by a reverse osmosis membrane which allows dewatering, resulting in supersaturated conditions which in turn cause nucleation and protein crystal growth in the mother liquor. The main advantage of this method is that the rate of dewatering can be determined by the difference in osmotic pressure on either side of the membrane. One drawback of this method is that the nucleation and subsequent protein crystal growth depends on increasing the concentration of precipitant and protein in the mother liquor. There is no control over the effects of solute driven convection on the surface of the crystal. As is the case with the protein crystals grown under conditions of microgravity, the crystals are not protected by any enclosure thus they are subject to physical damage as they are harvested and mounted. None of the existing methods for growing large, perfect crystals provide adequately protected protein crystals.
In conventional x-ray diffraction studies to elucidate the three-dimensional structure of a protein, in order to avoid physical damage to protein crystals, the crystals have typically been mounted in aqueous gels. There are problems, however, in removing the gel material without affecting the integrity of the protein crystal. It would be desirable if a protein crystal could be encapsulated in a shell or membrane that was able to protect the crystal from harsh environments which can cause degradation. A crystal contained within a closed, non-degrading environment would be useful to those working in fields requiring high quality, intact protein crystals. Also needed is a way to grow larger and better quality protein crystals by eliminating some of the physical factors which perturb crystal growth and by better controlling the dewatering conditions to promote single crystal growth. It would be desirable to have a method of preparing protein crystals entrapped in liquid filled microcapsules surrounded by a thin, flexible outer membrane, yet are sturdy enough to protect the enclosed crystals from conditions which might cause fracture or fluid convection that can alter the molecular arrangement at the crystal surface, or dissolution.
Also needed are better carriers for drugs, particularly crystalline drugs, which can resist prematurely rupturing and can provide sustained and/or controlled release at a therapeutic target site, and protect tissues from the sharp edges of the crystals.
Accordingly, it is an object of the present invention to provide microcapsules containing solutions and/or crystals of bioactive substances such as drugs and proteins, that have semi-permeable membranes which are rugged enough to protect fragile crystals and to resist shear and other mechanical forces typically associated with handling of such crystals.
It is another object of the present invention to provide microcapsules containing highly ordered structures of other bioactive agents, or biomolecules, such as DNA, RNA or oligonucleotides, which are capable of being transported intact through the human vascular system for release at a desired site of action.
It is another object of the invention to provide microcapsules having outer xe2x80x9cskinsxe2x80x9d or membranes which avoid being readily detected and eliminated by the reticuloendothelial system, and which protect the microcapsules against shear forces encountered during use, particularly during transport within the vascular system en route to target tissues.
Another object is to optimize the concentration of a bioactive agent in a microcapsule in order to achieve subsequent sustained or controlled release of the agent.
It is a further object of the invention to provide microcapsules which provide a closed environment that is favorable for growth of crystals under prescribed conditions of dewatering.
Still another object of the invention disclosed herein is to provide a method for making custom microcapsules containing protein crystals of suitable quality for X-ray diffraction studies of native and activated protein structures.
A further object of the invention to provide larger microcapsular packages containing saturated or near-saturated solutions of these bioactive substances than has been possible before, and to provide an environment for these microcapsules that is conducive to growing large crystals inside the microcapsule, or to accommodate larger 3-D ordered structures than has previously been possible.
By entrapping protein crystals in these special purpose microcapsules, they can be protected from conditions which might cause fracture of the crystals or fluid convection which can alter the molecular arrangement at the crystal surface, or dissolution. The semi-permeable membrane provided by the invention not only protects the crystals from harsh environments which can cause degradation, it also provides a closed environment which favors crystal growth under prescribed conditions of controlled dewatering.
In addition to crystallizable proteins or drug that are chemical compounds, many other bioactive substances which are capable of forming a highly ordered structure may be similarly microencapsulated. For instance, a duplex DNA strand or RNA-like structure, or a concentrated solution of an oligonucleotide or a polyribo- or -deoxyribonucleotide or other labile biological are also suitable for entrapment according to the methods of the present invention. Accordingly, the present invention provides a basic method of making a microcapsule comprising preparing a first phase containing a first solvent, a co-solvent and a first polymer dissolved therein. The method includes preparing a second phase of different density than the first phase, the second phase also including a second solvent, a surfactant, a salt, and a bioactive substance, all dissolved in the second phase. In this method the first polymer and surfactant are selected such that the hydrophobic/lipophilic balance value (HLB) of the surfactant is greater than the HLB of the first polymer. Thusly made, the first and second phases are capable of forming a mutual interface.
The basic method of making a microcapsule that contains a protein or bioactive agent preferably also has a second polymer dissolved in the second phase. In this case, the first polymer, second polymer and surfactant are each selected such that their respective hydrophobic/lipophilic balance values (HLB) are in the following order: surfactant greater than second polymer greater than first polymer. Upon bringing the two phases together gently, to form an interface, and by limiting fluid shear forces at the interface to about 0-50 dynes/cm2, microcapsules containing the dissolved bioactive agent are formed. Preferably the shear forces are limited to about 0-100 dynes/cm2 so as to form larger microcapsules.
In preferred embodiments of the invention, the bioactive substance is a protein which is dissolved in the second phase at a concentration that is at or near saturation. In some embodiments, a crystal of the protein is also suspended in the second phase solution. If the protein is particularly susceptible to degradation, a protein stabilizing agent may be included in the second phase solution.
The first solvent is preferably water, methanol, ethanol, isopropanol, n-hexanol, or n-heptanol, or a hydrocarbon having a low or medium HLB 5-10. The co-solvent is preferably a 3-carbon to 8-carbon (C3-C8) normal alcohol, tetrahydrofuran, dioxane, acetonitrile. dimethylformamide, dimethylacetamide, dimethylsulfoxide or a similar solvent.
The first polymer is preferably a polymer of glycerol monostearate, glycerol monooleate, glycerol monolaurate, glycerol dioleate, glycerol distearate or other hydrophobic mono- or polyglycerides or waxy polymers of low molecular weight, or it can be a combination of any of those polymers. In an alternative method of the invention, however, the first solvent is water and the first polymer is a polyethylene glycol having a molecular weight greater than about 400 kd, cyclodextrin, polyvinylpyrrolidine or polyvinyl alcohol. In another alternative embodiment, an alternative membrane forming material comprising a sterol or a phospholipid is substituted for the first polymer. The sterol or phospholipid may be cholesterol, stigmasterol, phytosterol, campesterol, phosphatydyl choline or CENTROLEX-F(trademark).
In preferred methods of making the microcapsules of the present invention, the second solvent is water and the surfactant has a hydrophilic/lipophilic balance value (HLB) of about 10-40. The HLB of the first polymer is preferably less than the HLB of the surfactant by 2 or more HLB units. The surfactant may be chosen from the group consisting of sorbitan monooleate plus ethylene oxide, dextran, polyethylene glycol (PEG), C12-C20 fatty acids, and quaternary NH4 salts.
The second polymer is preferably capable of adhering to the first polymer and is chosen from the group consisting of PEG 400-20000, dextran 4000-20,000, a polysaccharide of mol. wt. ranging from about 10,000-100,000, polyvinylpyrrolidone (PVP), a polyvinyl alcohol and other similar polymeric materials.
In preferred embodiments the salt contained in the second phase solution is NaCl, KCl, CaCl2, quaternary NH4 salts, cetyl trimethylammonium bromide, 2-amino-2-methyl aminomethyl propanol or a similar salt.
According to the preferred methods of the invention, after initial formation of the microcapsule, the membrane is allowed to cure. After curing, the relatively sturdy microcapsules may be separated into fractions of a certain size range, if desired for a particular purpose, such as injection into a blood vessel for therapeutic treatment. After curing, the microcapsules may then be subjected to gradual dewatering in order to gently bring about supersaturation of the bioactive agent and to encourage single crystal nucleation and growth. Optionally, an additional coating of polymer may be applied to the microcapsule, after curing, after dewatering, or after full growth of the crystal has been accomplished, in order to provide a thicker, more protective skin on the microcapsule.
The dewatering step of certain embodiments of the invention may include exposing the microcapsule to a closed local environment which is capable of regulating the rate and extent of microcapsule dewatering whereby controlled crystallization of a protein occurs within said microcapsule. The dewatering step may include exposing microcapsules to a dewatering solution containing a salt or a polymer which is excluded by the semi-permeable membrane of the microcapsule. In an alternative embodiment, the method includes diffusing a low molecular weight salt into said interior cavity to induce single crystal nucleation and crystal growth.
In certain embodiments employing a closed local environment for dewatering the microcapsule, the environment may also permit controlling the protein concentration and the concentration of charged precipitant molecules at or near the surface of a growing protein crystal so that the internal order and extent of crystallization of said protein crystal is optimized.
One preferred method of making a microcapsule includes preparing a first phase containing a first solvent chosen from the group consisting of: methanol, ethanol, isopropanol, m-hexanol, or n-heptanol, a co-solvent chosen from the group consisting of: a 3-carbon to 8-carbon (C3-C8) normal alcohol, and a first polymer dissolved in the first phase. The first polymer is a hydrophobic mono- or polyglyceride. According to this method, a second phase is also prepared, the second phase having a different density than that of the first phase. The second phase is water containing polyethylene glycol, as a surfactant, and a second polymer dissolved therein. This second polymer, which is capable of adhering to the first polymer, is PEG 1000-8000. A protein is also dissolved to saturation or near-saturation in the second phase. Optionally, one or more crystals may also be suspended in the second phase solution. The second phase also includes NaCl dissolved therein. An important feature of this method is that the first polymer, second polymer and surfactant are chosen such that the hydrophobic/lipophilic balance values (HLB) are: surfactant greater than second polymer greater than first polymer. When the two phases are gently brought into direct contact, an interface forms between them. It is a critical part of this method that the fluid shear stress at the interface be limited to 0-100 dynes/cm2 so that microcapsules having the desired characteristics will form. After microcapsules have formed, the outer membrane is then cured to make it more rugged and durable. Optionally, an additional polymer coating may be applied over the outer membrane if an even thicker membrane, or skin, is desired.
Also provided in accordance with the present invention is an improved method of determining the three-dimensional structure of a predetermined protein molecule by x-ray crystallography. The improvement includes forming a microcapsule containing a saturated or near saturated aqueous solution of a protein surrounded by a semi-permeable polymeric membrane. The microcapsule is exposed to a dewatering solution having a higher osmotic pressure than the encapsulated protein solution, whereby water is osmotically removed from said encapsulated protein solution. By controlling the concentration of a dewatering agent in the dewatering solution, gradual, ordered crystallization of the protein occurs within the microcapsule. This gradual, ordered crystal growth is allowed to continue until the crystal becomes at least about 50-300 microns across one face. A microcapsule containing a crystal of sufficient size and crystalline quality is then carefully selected. The microcapsule is mounted in an x-ray capillary tube and subjected to a high energy x-ray crystallographic procedure to obtain a characteristic x-ray diffraction pattern of the protein crystal.
In an alternative and preferred method of performing x-ray crystallography on a crystal specimen, the present invention provides an improvement over methods which include isolating a crystal specimen in a fiber loop with an attached handle portion, freezing the crystal specimen, mounting the crystal specimen and fiber loop on a goniometer head such that said crystal is positioned in a continuous N2 stream loop, and rotating the goniometer head in an x-ray beam. The present improvement includes substituting for the conventional crystal specimen a microencapsulated crystal that has a protective outer membrane surrounding a large crystal and a small amount of mother liquor. The membrane, which is preferably a composite of two or more polymers, is substantially transparent to the x-ray beam so that it does not interfere with the x-ray diffraction pattern of the crystal. According to this improved method, the membrane has an electrostatic charge which renders the microencapsulated crystal electrostatically attracted to the fiber loop. This electrostatic attraction is strong enough to support the microencapsulated crystal inside said loop. Optionally, a drop of liquid may be adhered to the outer membrane of the microencapsulated crystal to facilitate freezing. In certain embodiments the membrane is negatively charged and the loop is a fiber having a positive electrostatic charge. In certain preferred embodiments the crystal is a highly ordered protein crystal.
The present invention also provides a microencapsulated protein crystal prepared by certain methods described above. In some embodiments the microcapsule is best characterized as a product of a particular method of the invention, because the inventors believe that there are as yet unrealized features and characteristics of the new microcapsules which are attributable to the novel method of making.
In accordance with the present invention, a microcapsule is provided having an outer membrane surrounding an interior cavity, the interior cavity containing a saturated or nearly saturated solution of a bioactive agent. An important feature of this new microcapsule is that it is capable of withstanding shear forces at least as great as the turbulent blood flow within a human artery.
Certain embodiments of the new microcapsule have a membrane containing at least one of the membrane forming material materials described above in the summary of the methods. descriptions. In the preferred embodiments, the membrane is a composite containing a first polymer and a second polymer that is capable of adhering to the first polymer. The HLB of the second polymer is preferably greater than the HLB of the first polymer.
In certain embodiments of the microcapsule of the present invention, the interior cavity also contains the protein or bioactive agent in the form of a highly ordered structure such as a crystal. In some embodiments the crystal substantially fills the interior cavity. The membrane may even substantially conform to the shape of a large crystal. In preferred embodiments, the membrane is resistant to rupturing or piercing by the crystal.
In certain embodiments the microcapsule""s membrane is permeable to water and low molecular weight salts but impermeable, or only slightly permeable, to the bioactive agent. In some embodiments, the membrane is less than or equal to I micron in thickness, and in others the membrane is about 3-5 microns thick.
Preferably the bioactive agent is a protein or a drug, however in some embodiments of the microcapsule of the invention the bioactive agent is a biomolecule such as a polypeptide, oligonucleotide. RNA, DNA or other compound which can be crystallized.
Certain embodiments of the new microcapsule include a highly ordered structure, such as a crystal, about 50-2000 microns in size.
Certain alternative embodiments of the microcapsule of the invention have an interior cavity that contains a hydrophobic phase surrounded by and partially immiscible with a saturated or near-saturated solution of the bioactive agent.
Also provided by the present invention is a composition comprising a multiplicity of certain microcapsule of the invention suspended in an aqueous solution having higher osmotic pressure than that of the bioactive agent solution. The higher osmotic aqueous solution may include a dewatering agent capable of causing water to be transported through said membrane and out of said interior cavity. This dewatering agent may be a salt or a high molecular weight polymer which is excluded by said membrane.
Certain embodiments of the new microcapsules have a polymeric membrane that is transparent to x-ray radiation and/or does not interfere with the x-ray diffraction pattern of the highly ordered structure.
The present invention accordingly provides an x-ray crystallography reagent for use in elucidating the three-dimensional structure of a predetermined biomolecule which is capable of forming a highly ordered structure. The reagent comprises a dewatered microcapsule prepared according to certain methods of the invention and having a highly ordered structure, such as a protein crystal, substantially filling the interior cavity of the microcapsule.
The present invention also provides a pharmaceutical composition comprising a pharmacologically effective multiplicity of certain microcapsules of the invention, together with a pharmacologically acceptable carrier. For particular medical uses, the average size of the microcapsules is about 1-20 microns, and for others the average size of said microcapsules is about 50-300 microns, or even greater than about 300 microns.